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Acinetobacter baumannii OxyR regulates the transcriptional response to hydrogen peroxide 1

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Lillian J. Juttukonda1, Erin R. Green

1, Zachery R. Lonergan

1, Marie C. Heffern

2,3, Christopher J. 3

Chang3,4,5

, and Eric P. Skaar1# 4

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1Vanderbilt Institute for Infection, Immunology, and Inflammation and Department of 6

Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, 7

TN 37232, USA 8

2Present address: Department of Chemistry, University of California, Davis, CA 95616, USA. 9

3Department of Chemistry, University of California, Berkeley, CA 94720, USA. 10

4Department of Molecular and Cell Biology University of California, Berkeley, CA 94720, USA. 11

5Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA. 12

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#Address correspondence to: 14

Eric P. Skaar 15

Vanderbilt University School of Medicine 16

Department of Pathology, Microbiology and Immunology 17

MCN A-5301 18

Nashville, TN 37232-2363 19

Phone: 615-343-0002 20

Fax: 615-343-7492 21

E-mail: [email protected] 22

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IAI Accepted Manuscript Posted Online 8 October 2018Infect. Immun. doi:10.1128/IAI.00413-18Copyright © 2018 American Society for Microbiology. All Rights Reserved.

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Running title: OxyR regulon in Acinetobacter baumannii 24

Keywords: Hydrogen peroxide, Acinetobacter baumannii, OxyR, in vivo imaging, transcriptional 25

regulation, pneumonia, RNA sequencing 26

Conflict of interest statement: The authors have declared no conflicts of interest. 27


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Abstract 28

Acinetobacter baumannii is a Gram-negative opportunistic pathogen that causes diverse 29

infections, including pneumonia, bacteremia, and wound infections. Due to multiple intrinsic and 30

acquired antimicrobial-resistance mechanisms, A. baumannii isolates are commonly multi-drug 31

resistant and infections are notoriously difficult to treat. The World Health Organization recently 32

highlighted carbapenem-resistant A. baumannii as a ‘critical priority’ for the development of new 33

antimicrobials because of the risk to human health posed by this organism. Therefore, it is 34

important to discover mechanisms used by A. baumannii to survive stresses encountered during 35

infection in order to identify new drug targets. In this study, we identified hydrogen peroxide 36

(H2O2) as a stressor produced in the lung during A. baumannii infection using in vivo imaging 37

and defined OxyR as a transcriptional regulator of the H2O2 stress response. Upon exposure to 38

H2O2, A. baumannii differentially transcribes several hundred genes. However, transcriptional 39

upregulation of genes predicted to detoxify hydrogen peroxide is abolished in A. baumannii 40

genetically inactivated for the transcriptional regulator oxyR. Moreover, inactivation of oxyR in 41

both antimicrobial-susceptible and multi-drug-resistant A. baumannii strains impairs growth in 42

the presence of H2O2. OxyR is a direct regulator of katE and ahpF1, which encode the major 43

H2O2-degrading enzymes in A. baumannii, as confirmed through measurement of promoter 44

binding by recombinant OxyR in electromobility shift assays. Finally, an oxyR mutant is less fit 45

than wild-type A. baumannii during infection of the murine lung. This work reveals a mechanism 46

used by this important human pathogen to survive H2O2 stress encountered during infection. 47

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Introduction 49

Acinetobacter baumannii is a Gram-negative coccobacillus and an obligate aerobe. A. 50

baumannii is most well-known as an agent of ventilator-associated pneumonia and bloodstream 51

infections in critically ill patients but also causes hospital-acquired infections of virtually any 52

body site, including skin and soft-tissue infections, wound infections, urinary tract infections, 53

and meningitis (1, 2). Since the original recognition of A. baumannii as a frequent opportunistic 54

pathogen, this organism has emerged as one of the most difficult bacterial infectious agents to 55

treat because of extensive intrinsic and evolved antimicrobial resistance (1, 3-6). Globally, 63% 56

of A. baumannii isolates were found to be multidrug resistant in 2014, an increase from 23% in 57

2004 (7). In particular, the threat to global human health from increasing rates of carbapenem-58

resistant A. baumannii infections has led the World Health Organization to name carbapenem-59

resistant A. baumannii the number one ‘critical’ priority for the development of new antibiotics 60

(8). Therefore, research investigating mechanisms by which A. baumannii survives within hosts 61

and in the environment is urgently needed as a strategy to identify new targets for therapeutic 62

intervention. 63

A. baumannii does not typically use classical virulence determinants such as effector 64

toxins or immune evasion strategies (3, 9). Instead, A. baumannii utilizes genes evolved for 65

survival in the environment to persist during infection (9, 10). Moreover, the ability of A. 66

baumannii to survive within the hospital environment permits this organism to cause outbreaks 67

of hospital-associated infections that are difficult to eradicate (11). We therefore predict that 68

processes involved in sensing and responding to the environment are critical for A. baumannii to 69

be a successful opportunistic pathogen. 70


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During infection, bacteria experience numerous stresses imposed by the host. One stress 71

thought to be encountered during infection is reactive oxygen species produced by effector cells 72

of the innate immune system (12). Neutrophils treated with various pro-inflammatory stimuli ex 73

vivo generate an oxidative burst initiated by NADPH phagocyte oxidase that results in the 74

production of superoxide, H2O2, hypochlorous acid and peroxynitrate (12). Mice lacking 75

NADPH phagocyte oxidase are more susceptible to A. baumannii pneumonia (13). Therefore, we 76

hypothesized that H2O2 is formed in the lung during A. baumannii pneumonia and thus is a stress 77

that A. baumannii must overcome in order to survive and infect the lung. 78

Bacteria have evolved mechanisms to sense damaging molecules and induce an 79

appropriate transcriptional response, such as upregulation of enzymes that detoxify reactive 80

oxygen species. Transcriptional regulators that sense either the reactive molecule itself or the 81

damage caused by reactive molecules control the production of detoxification proteins (14). 82

H2O2 is detoxified by alkyl hydroperoxide reductase (Ahp) and catalase (Kat), which reduce 83

H2O2 to water (15). In A. baumannii, the catalase genes katE and katG and the universal stress 84

protein UspA protect against H2O2 stress (16, 17). However, regulatory mechanisms that govern 85

the transcriptional response of A. baumannii to H2O2 have not been characterized. 86

In Gram-negative bacteria, OxyR is the canonical orchestrator of the H2O2 detoxification 87

response (18). OxyR is a highly conserved transcriptional regulator of the LysR-family that 88

senses and responds to H2O2 stress (19-21). Oxidation of a conserved cysteine residue causes 89

OxyR to undergo a conformational change, resulting in altered DNA binding (21-25). In 90

Escherichia coli, OxyR serves as a transcriptional activator of genes involved in H2O2 91

detoxification, including ahp which encodes alkyl hydroperoxide reductase, kat which encodes 92

catalase, and dps which encodes DNA protection during starvation protein (19, 26). While OxyR 93


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is highly conserved, the function of this protein varies between organisms. For instance, while 94

OxyR is classically described as a transcriptional activator, OxyR in Corynebacterium 95

glutamicum is a transcriptional repressor, and inactivation of oxyR enhances resistance to H2O2 96

in this organism (27). There are also discrepancies regarding the sensitivity of oxyR mutants to 97

H2O2 killing during log phase growth. Xanthomonas campestris lacking oxyR is sensitive to 98

H2O2 during log phase growth but Neisseria meningitidis and Brucella abortus inactivated for 99

oxyR are resistant to H2O2 killing in exponential growth (28-30). While oxyR homologues in A. 100

baumannii and other Acinetobacter species have been identified and shown to confer resistance 101

to H2O2 in agar plates, the functional role of A. baumannii OxyR in transcriptional regulation and 102

survival in vivo has not been studied (31-33). 103

We hypothesized that A. baumannii encounters H2O2 stress during infection and that 104

OxyR controls the transcriptional response to this stress. In this study, we demonstrate that H2O2 105

is formed during A. baumannii infection of the lung and characterize the transcriptional response 106

of A. baumannii to H2O2 by RNA sequencing. Furthermore, we define the role of A. baumannii 107

oxyR in defense against H2O2 stress, determine the regulon of OxyR by RNA sequencing, and 108

confirm direct promoter binding by recombinant OxyR. Finally, we study the role of oxyR in A. 109

baumannii infection of the murine lung. By identifying the role and regulon of OxyR in A. 110

baumannii, we have defined a transcriptional regulatory network in this important human 111

pathogen that confers resistance to stresses encountered by this bacterium during infection. 112

These findings provide a foundation for future development of novel therapeutics against A. 113

baumannii infections. 114

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Results 116


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A. baumannii lung infection induces H2O2 production 117

H2O2 is produced by the neutrophil oxidative burst (12), and neutrophils are recruited in 118

response to A. baumannii infection (34). In order to test our hypothesis that H2O2 functions as an 119

antimicrobial during A. baumannii lung infection, we first wanted to determine if we could 120

directly visualize H2O2 production in vivo. Thus, we employed a recently developed 121

bioluminescence system in which a caged luciferin probe specifically reacts with H2O2 to release 122

D-luciferin, which leads to bioluminescence in mice that constitutively express luciferase 123

(Figure 1A) (35, 36). Upon intranasal inoculation with A. baumannii ATCC 17978, substantial 124

bioluminescence was detected in the thoracic cavity, whereas minimal bioluminescence was 125

detected following mock-infection with PBS (Figure 1B & C). Based on these data, we 126

conclude that infection with A. baumannii stimulates the production of H2O2 in the lung. 127

H2O2 exposure alters the transcriptome of A. baumannii in an oxyR-dependent manner 128

Considering the success of A. baumannii as a pathogen, this organism must survive an 129

onslaught of H2O2 produced in vivo. The mechanisms by which A. baumannii defends against 130

H2O2 are not defined. We characterized the response of A. baumannii to H2O2 exposure by RNA 131

sequencing. Compared to the mock-treated group, cells exposed to H2O2 upregulated the 132

transcription of 155 genes and downregulated the transcription of 151 genes (Figure 2A & B; 133

Table 2 & Supplemental Table 2). Genes encoding proteins that directly detoxify H2O2 were 134

strongly upregulated, including ahpF1 (A1S_1200-1201), ahpF2 (A1S_1458-1460), and katE 135

(A1S_3382). Genes involved in sulfur transport and iron homeostasis were also highly 136

upregulated by H2O2 exposure, which we postulate is in response to oxidation of iron-sulfur (Fe-137

S) clusters by H2O2 (15). Amino acid transport and metabolism pathways were also upregulated 138

by H2O2, perhaps as a repair or replacement mechanism for proteins that sustained oxidative 139


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damage. A. baumannii also increases transcription of phosphate transport and nucleic acid 140

synthesis and repair genes, consistent with H2O2-induced DNA damage (15). Treatment with 141

H2O2 predominantly downregulates metabolic and synthetic genes, suggesting a model in which 142

most cellular metabolism is paused while the acute toxicity is managed (Supplemental Table 2). 143

Many Gram-negative organisms defend against H2O2 by inducing the OxyR regulon, 144

which includes genes encoding the H2O2-reducing enzymes catalase and alkyl hydroperoxide 145

reductase (19, 26, 37). As A. baumannii is known to encode an OxyR homologue (33), we 146

hypothesized that OxyR is important for the transcriptional changes induced by H2O2. To test 147

this hypothesis, an A. baumannii mutant (oxyR) was generated which contained an in-frame 148

replacement of oxyR (A1S_0992) with the kanamycin resistance cassette aph1. The 149

transcriptional profile of the oxyR strain in the presence and absence of H2O2 was characterized 150

by RNA sequencing (Figure 2B, Table 3 & Supplemental Table 3). Consistent with our 151

hypothesis, only 15 genes were upregulated by H2O2 in oxyR, demonstrating that oxyR is 152

important for activating the transcriptional response to H2O2. To validate the RNA sequencing 153

results, qRT-PCR was performed on selected genes that exhibited the most significant changes in 154

expression in the RNASeq data (Figure 2C). Transcript abundance of A1S_1200 (ahpF1), 155

A1S_1458 (ahpF2), A1S_2459 (oxidoreductase), and A1S_2531 (sulfate transporter) were 156

significantly increased following H2O2 treatment in an oxyR-dependent manner, while transcript 157

abundance of A1S_0104 (acyl-CoA synthesis) and A1S_2150 (oxidoreductase) were significantly 158

decreased in an oxyR-dependent manner. Together, these results demonstrate that in A. 159

baumannii, OxyR is an important global regulator of the transcriptional response to H2O2. 160

Inactivation of oxyR impairs A. baumannii growth in the presence of H2O2 161


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Based on the inability of the oxyR strain to upregulate detoxification genes upon 162

exposure to H2O2, we hypothesized that the oxyR strain would have a growth deficiency in 163

medium containing H2O2. The A. baumannii oxyR strain did not have a significant growth 164

defect in LB and, in fact, grew to a higher cell density in stationary phase (Supplemental Figure 165

1A). However, when grown in the presence of 100 M H2O2, the oxyR strain lag time was 6 166

hours longer than for WT (Figure 3A). The growth lag of the oxyR strain in H2O2 was 167

complemented by expressing oxyR in trans under the control of a constitutive promoter 168

(Supplemental Figure 1B). Growth of the oxyR strain was impaired by concentrations of H2O2 169

as low as 50 M (Figure 3B). Similarly, in a H2O2 disc diffusion assay, the oxyR strain 170

exhibited a substantially greater zone of inhibition by H2O2 than WT (Figure 3C&D). 171

A gene encoding OxyR was present in every annotated A. baumannii genome, suggesting 172

that its regulatory role is conserved within the species (Supplemental Table 4). We evaluated 173

the role of oxyR in growth of the A. baumannii strain AB5075, a multidrug-resistant clinical 174

isolate. AB5075 mutant strains with transposons within the oxyR allele (ABUW_2905) were 175

obtained from the University of Washington sequenced transposon library (38). Four 176

independent ABUW_2905::tn mutants were evaluated for growth in LB in the absence or 177

presence of H2O2. In contrast to the 17978 oxyR mutant, AB5075 ABUW_2905::tn mutants 178

exhibited impaired growth in LB (Supplemental Figure 1C). Decreased growth in the absence 179

of exogenous H2O2 has been previously observed for oxyR mutants in other organisms (39). In 180

the presence of H2O2, ABUW_2905::tn mutants are strikingly impaired for growth 181

(Supplemental Figure 1D&E). Taken together, these results demonstrate that oxyR is important 182

for growth of multiple A. baumannii strains in the presence of H2O2. 183

A conserved cysteine residue is important for OxyR function 184


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A phylogenetic analysis comparing A. baumannii OxyR to other characterized OxyR 185

proteins revealed that A. baumannii OxyR is in the same clade as OxyR from other 186

Proteobacteria (Figure 4A). In E. coli, OxyR senses H2O2 by a hyperreactive thiol in a conserved 187

cysteine residue (C199) that is rapidly oxidized by H2O2 (37). Mutational analysis in E. coli 188

identified that mutation of this cysteine residue to serine locks OxyR in the reduced state (40). 189

Alignment of the primary amino acid sequence revealed that C202 in A. baumannii OxyR aligns 190

with C199 in E. coli (Figure 4B). Therefore, we hypothesized that C202 is important for the 191

function of A. baumannii OxyR. Expression of oxyR with cysteine 202 mutated to serine in trans 192

did not complement the growth defect of the oxyR strain in H2O2 (Figure 4C). These data 193

suggest that this conserved cysteine residue is critical for the function of OxyR in A. baumannii. 194

A. baumannii oxyR is pre-adapted to H2O2 stress 195

We next investigated whether OxyR altered survival of A. baumannii cultures in a H2O2 196

killing assay. As expected, increasing concentrations of H2O2 resulted in decreased survival of 197

WT A. baumannii (Figure 5A). However, the oxyR strain was resistant to killing by H2O2 in 198

these conditions, with no decrease in survival until 40 mM H2O2 was applied (Figure 5A). Based 199

on these results, we hypothesized that the oxyR strain is protected from H2O2 killing because 200

this strain has altered expression of peroxide detoxification machinery in LB. To test this 201

hypothesis, the RNASeq data were analyzed to compare the transcriptional profile of WT A. 202

baumannii and the oxyR strain in LB (Table 4 & Supplemental Table 5). Consistent with our 203

model, the oxyR strain expressed higher levels of ahpF1 (A1S_1200-1201), ahpF2 (A1S_1458-204

1460), and katE (A1S_3382) in LB, which was confirmed by qRT-PCR (Figure 5B). Because 205

these genes are also induced by H2O2 exposure, we posited that low-dose H2O2 exposure adapts 206

A. baumannii to H2O2 stress, protecting against killing by higher concentrations of H2O2. Indeed, 207


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pre-treatment with 1 mM H2O2 enhanced survival of WT A. baumannii following high dose 208

H2O2 stress (Figure 5C). However, pre-treatment of the oxyR strain with 1 mM H2O2 did not 209

alter survival of the oxyR strain, suggesting that the oxyR strain is pre-adapted to H2O2 stress 210

(Figure 5C). Together, these results reveal that A. baumannii adapts to H2O2 stress and the 211

oxyR strain is phenotypically pre-adapted to H2O2 stress and resistant to H2O2 killing. This 212

result is consistent with the finding that ahpF1 (A1S_1200-1201), ahpF2 (A1S_1458-1460), and 213

katE (A1S_3382) expression does not increase with H2O2 treatment in the oxyR strain (Figure 214

2C) but expression is high in the absence of H2O2 (Figure 5B). Mechanistically, these data 215

suggest that OxyR functions as a repressor of the ahpF1 (A1S_1200-1201), ahpF2 (A1S_1458-216

1460), and katE (A1S_3382) genes, as the oxyR strain overexpresses these genes. 217

OxyR binds to the promoters of ahp and kat 218

We hypothesized that OxyR directly binds to the promoter regions of genes encoding 219

H2O2 detoxification machinery. To determine if OxyR directly binds the promoter regions of 220

putative target genes in a H2O2-dependent manner, electromobility shift assays (EMSAs) were 221

carried out with OxyR bearing an N-terminal hexahistidine tag (Supplemental Figure 2A). 222

OxyR was purified under reducing conditions, and promoter binding was tested in the presence 223

or absence of excess H2O2. OxyR binds the promoter regions of A1S_1201 (ahpF1) and 224

A1S_3382 (katE), and binding occurs with lower concentrations of OxyR following incubation 225

with H2O2 (Figure 6A & B). To test the hypothesis that the cysteine at position 202 is important 226

for regulation of DNA binding activity, binding of recombinant OxyR C202S to the promoter 227

region of A1S_1201 (ahpF1) was assessed. In the presence of H2O2, a DNA mobility shift was 228

present at lower concentrations of protein for WT OxyR than for OxyR C202S, suggesting that 229

in these conditions C202 is important for DNA binding activity (Figure 6C). Interestingly, under 230


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reducing conditions, OxyR C202S had similar DNA binding activity to WT OxyR, suggesting 231

that OxyR C202S is not important for DNA binding activity in the absence of H2O2 (Figure 6D). 232

In addition, OxyR C202S was capable of binding the promoter region of A1S_3382 (katE) 233

similarly to WT OxyR under reducing conditions, but demonstrated decreased binding in the 234

presence of H2O2 (Supplemental Figure 2B). WT OxyR and OxyR C202S did not cause DNA 235

shifts of the negative control promoter mumT (Supplemental Figure 2C). Together, these data 236

demonstrate that A. baumannii OxyR is a DNA-binding protein that exhibits differential DNA 237

binding ability for target genes following exposure to H2O2. Furthermore, C202 modulates 238

binding to target DNA promoters in the presence of H2O2 but not in the absence of H2O2. The 239

fact that OxyR binds to ahpF1 and katE in the absence of H2O2 is consistent with the model that 240

A. baumannii OxyR is a repressor of these genes in the absence of H2O2. 241

oxyR promotes the fitness of A. baumannii in a murine model of pneumonia 242

As H2O2 is produced in the lung during A. baumannii pneumonia and OxyR orchestrates 243

the transcriptional response to H2O2 in vitro, we hypothesized that OxyR promotes fitness of A. 244

baumannii during infection of the murine lung. Mice were co-infected intranasally with A. 245

baumannii WT and the oxyR strain and bacterial burdens were enumerated 36 hours post-246

infection. The oxyR strain was recovered at significantly lower numbers than WT A. baumannii 247

in the lung (Figure 7A), indicating that WT is more fit than the oxyR strain. The oxyR strain 248

was also less able to disseminate to the liver (Figure 7B). Therefore, oxyR promotes the fitness 249

of A. baumannii during infection. 250

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Discussion 252


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We report here that A. baumannii encounters H2O2 during infection of the murine lung 253

and employs a sophisticated strategy to survive and grow in the presence of H2O2. 254

Transcriptional profiling of A. baumannii exposed to H2O2 revealed that the canonical oxyR 255

regulon is induced by H2O2 in an oxyR-dependent manner. A. baumannii lacking oxyR has 256

impaired growth in the presence of H2O2, but the oxyR strain overexpresses ahp and katE 257

during aerobic growth and is protected from H2O2-mediated killing. We demonstrate that A. 258

baumannii OxyR contains a functionally important conserved cysteine residue and exhibits 259

H2O2-dependent promoter binding activity. Finally, oxyR confers a fitness advantage in a 260

competitive murine model of pneumonia. 261

People who lack an effective neutrophil oxidative burst due to mutations in NADPH 262

oxidase develop frequent and severe infections from catalase-producing organisms, emphasizing 263

the importance of the neutrophil oxidative burst in protection against infection (41). While the 264

production of H2O2 by neutrophils can be monitored easily in cell culture assays (42), visualizing 265

the neutrophil oxidative burst that occurs during infection is a technical challenge because 266

reactive oxygen and nitrogen species are highly reactive by definition. Here we used a recently 267

described chemical probe to detect in vivo H2O2 production in real time in a live animal. We 268

found that infection of the lung with A. baumannii instigated H2O2 production specifically within 269

the thoracic cavity, illustrating the harsh environment that A. baumannii must endure to 270

successfully colonize the lung and cause disease. 271

RNA sequencing of A. baumannii exposed to H2O2 revealed a robust transcriptional 272

response that shared some transcriptional changes with previously published RNA sequencing 273

and microarray experiments in other bacteria (43-54). Interestingly, RNA sequencing revealed 274

that H2O2 exposure leads to the downregulation of many metabolic genes, including the paa 275


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operon, which has been shown to enhance the virulence of A. baumannii in a murine sepsis and 276

zebrafish infection model (55, 56). Analysis of the RNA sequencing data provides insight into 277

genes that may be activated by OxyR in the presence of H2O2. Compared to the well-studied 278

OxyR regulon in E. coli (57), A. baumannii shares upregulation of ahpF1, ahpF2, and catalase, 279

but not other genes known to be regulated by E. coli OxyR, including fur, hemH, trxB, trxC, 280

gorA, or grxA. Thus, while OxyR is highly conserved amongst Gram-negative bacteria, the 281

OxyR regulon is not conserved across the Gammaproteobacteria, highlighting the functional 282

diversity of this regulator. 283

A. baumannii oxyR shares characteristics with oxyR mutants in multiple organisms. 284

Inactivation of oxyR in A. baumannii increases the zone of inhibition by H2O2 in a disc diffusion 285

assay, similar to B. abortus, Pseudomonas aeruginosa, Moraxella catarrhalis, and E. coli (28, 286

58-60). Elucidating the contribution of oxyR to H2O2 stress resistance adds to existing knowledge 287

of genes that protect against H2O2 toxicity in A. baumannii, including uspA, katE, and katG (16, 288

17). 289

In addition to its role in coordinating the response to exogenous H2O2, OxyR may also be 290

important in the context of endogenous H2O2. Indeed, in A. baumannii strain AB5075, 291

inactivation of oxyR leads to decreased growth rate in the absence of exogenously added H2O2. 292

In contrast, inactivation of oxyR in A. baumannii strain ATCC 17978 leads to higher optical 293

density of stationary phase cultures. One potential explanation for the finding the oxyR mutants 294

have growth phenotypes in the absence of exogenous H2O2 is that A. baumannii OxyR may be 295

important for protecting against endogenously produced H2O2, as has been shown in E. coli, 296

where OxyR induces H2O2-scavenging enzymes in response to phenylethylamine oxidase-297

generated H2O2 (61). However, another possible explanation of these data is that OxyR plays a 298


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role in fundamental cellular metabolism. Loss of oxyR reduces the efficacy of oxygen respiration 299

in Shewanella oneidensis and increased transcription of cytochrome bd (39); while there were no 300

significant differences in cyd operon expression noted in the RNA sequencing of the A. 301

baumannii oxyR strain in LB, it is possible that other aspects of central metabolism are altered 302

in this strain. There remains much to be learned about the function of OxyR in pathogenic 303

organisms, including its potential role in central metabolism. For instance, all Mycobacterium 304

tuberculosis isolates carry inactivated oxyR alleles (62), suggesting that oxyR may be detrimental 305

in some infectious niches and that the biological role of this protein remains incompletely 306

understood. Exploring central metabolism using oxyR-null strains may provide insight into this 307

biology. 308

Similar to B. abortus and N. meningitidis (28, 63), the A. baumannii oxyR strain is 309

resistant to H2O2 in exponential phase. This resistance correlates with the increased transcription 310

of katE, ahpF1, and ahpF2 in the oxyR strain in the absence of H2O2. There are at least two 311

possible models to explain upregulation of ahpF1, ahpF2, and katE in the oxyR mutant. The first 312

model is that loss of oxyR induces upregulation of oxyR-independent pathways that lead to 313

increased transcription of hydrogen peroxide detoxification genes. The second model is that 314

OxyR has a dual function as both a repressor and an activator of ahpF1, ahpF2, and katE 315

depending on the oxidation state of OxyR, which has been previously proposed for OxyR in 316

other organisms, including N. meningitidis and S. oneidensis (63, 64). In this dual repressor-317

activator model, reduced OxyR binding to the promoter represses transcription and oxidized 318

OxyR binding to the promoter activates transcription. Based on this hypothesis, loss of oxyR 319

would lead to de-repression of ahpF1, ahpF2, and katE in the absence of hydrogen peroxide and 320

thus an increase in the basal level of transcription. We propose that the A. baumannii oxyR 321


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strain is resistant to H2O2 killing because of the higher transcription of katE, ahpF1, and ahpF2 322

in the absence of H2O2, and increased transcription of the detoxification machinery provides 323

resistance to bolus dosing of H2O2. It would be interesting to test the hypothesis that the 324

increased resistance to H2O2 in the oxyR deletion strain is due to upregulation of ahpF1, ahpF2, 325

and katE through the generation of double, triple, or quadruple mutant strains, although this 326

undertaking would be technically challenging and analysis of the combination mutants would not 327

be straightforward as each mutant is likely to have a phenotype independently. Taken together, 328

these results highlight the importance of OxyR as a transcriptional regulator in both reduced and 329

oxidative environments. 330

This work provides evidence that A. baumannii OxyR functions as both a transcriptional 331

repressor and as a transcriptional activator. H2O2 induces expression of OxyR gene targets, 332

including ahpF1 and katE, in an OxyR-dependent manner. This result suggests that OxyR is an 333

activator. However, ahpF1 and katE have higher expression in the oxyR strain in the absence of 334

exogenous H2O2, consistent with OxyR repressing at these promoters. OxyR binds the ahpF1 335

and katE promoter regions with greater affinity following incubation with H2O2, consistent with 336

a mechanism of OxyR activation by oxidation of C202, but binding is not abolished in the 337

absence of H2O2. The finding that OxyR C202S is capable of binding the promoter of ahpF1 and 338

katE under reducing conditions, but binds less well under oxidizing conditions, is consistent with 339

the model that this residue is important for regulating DNA binding in the presence of H2O2. 340

Therefore, we propose that OxyR binds to these promoters in the absence of signal and represses 341

transcription. Following activation by H2O2, OxyR undergoes a conformational change and 342

activates transcription of the target genes. This dual repressor/activator function has been 343

previously described for regulation of the ahpC promoter by OxyR in X. campestris (65). 344


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However, another possible explanation for increased expression of ahpF1, ahpF2, and katE in 345

the absence of oxyR is that another transcriptional regulator can be activated by endogenous 346

peroxide when OxyR is absent. 347

Previous studies in E. coli have demonstrated that sensing of H2O2 requires a conserved 348

cysteine residue at 199. Oxidation of C199 results in a significant structural change to the 349

regulatory domain of E. coli OxyR (22). In this work, we demonstrate that C202 is important for 350

function of A. baumannii OxyR. Interestingly, the oxyR deletion strain complemented with oxyR 351

C202S grows more poorly in the presence of H2O2 than that oxyR deletion strain alone. We 352

expect that the OxyR C202S strain is “locked” in the reduced form, as was previously 353

demonstrated for E. coli OxyR C199S (22, 66). Therefore, the presence of reduced OxyR 354

appears to have a detrimental impact on A. baumannii growth in the presence of H2O2. As OxyR 355

C202S is capable of DNA binding, we hypothesize that this mutant causes dysregulated 356

expression of the OxyR regulon in the presence of H2O2, leading to impaired growth. 357

Finally, we report that oxyR increases the fitness of A. baumannii in a murine pneumonia 358

model. Importantly, this results corroborates the imaging data demonstrating that H2O2 is 359

produced in vivo during A. baumannii infection. The finding that oxyR contributes to A. 360

baumannii pathogenesis builds on work in P. aeruginosa, E. coli, Francisella tularensis, 361

Klebsiella pneumonia, and Bacteroides fragilis demonstrating that oxyR mutants are less fit in 362

vivo (67-71). These results define oxyR as a factor in A. baumannii that is important for fitness 363

during infection and contribute to understanding the pathogenesis of this organism, a critical 364

multi-drug resistant threat to human health. 365

366

Materials and Methods 367


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Bacterial strains and reagents. The strains used in this study are described in Table 1. Unless 368

otherwise noted, all strains are derivatives of the human clinical isolate A. baumannii ATCC 369

17978. Transposon mutants in the A. baumannii AB5075 background were purchased from the 370

University of Washington A. baumannii mutant library (38). Transposon insertion sites were 371

confimed by PCR amplification between transposon-specific primer Pgro-172 and primers 372

external to the oxyR allele (ERG19 for 5’ and ERG24 for 3’) (See Supplemental Table 1). 373

Cloning was performed in E. coli DH5α. Bacteria were routinely grown in lysogeny broth (LB) 374

at 37°C unless otherwise noted. Solid medium contained 1.5% agar. Antibiotics were added at 375

the following concentrations: 75 μg mL-1

carbenicillin and 40 μg mL-1

kanamycin. All antibiotics 376

were purchased from Sigma (St. Louis, MO). 377

Strain generation. Primers used in this study are listed in Supplemental Table 1. In-frame 378

deletion strain ΔoxyR was generated via one-step recombineering as previously described (33). 379

Briefly, the kanamycin resistance cassette aph1 from pUC18K1 was amplified using primer pairs 380

LEJ_209 and LEJ_220, which contained 120 bp regions of homology flanking the oxyR ORF. 381

The PCR product was purified using column PCR purification (QIAGEN, Hilden, DE), 382

concentrated to >1 g/L, and electroporated into WT A. baumannii containing pAT02 (33). 383

RecAb expression was induced with 2 mM isopropyl -D-1-thiogalactopyranoxide (IPTG), 384

cultures were grown at 37C for four hours, and half of the transformed cells were plated onto 385

LB agar containing kanamycin. Kanamycin resistant colonies were re-streaked onto LB agar 386

with kanamycin and screened by PCR for replacement of the oxyR allele with aph1 with primer 387

pairs LEJ_213 and LEJ_214. Clones with the correct insertion were restreaked to solid medium 388

containing kanamycin or carbenicillin to screen for the loss of the pAT02 plasmid. KanR carb

S 389

clones were saved at -80C. The oxyR complementation vectors was constructed in pWH1266 390


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under the control of the 16S promoter (r01) by PCR amplification of the open reading frame 391

(ORF), double digest of the vector and PCR product with BamHI-HF and SalI-HF (New England 392

Biolabs, Ipswich, MA), and ligation with T4 DNA ligase (Promega, Madison WI). Site-directed 393

mutagenesis was performed by subcloning the oxyR ORF into pCRBlunt (Invitrogen, Carlsbad, 394

CA) and amplifying the vector with Pfu Turbo polymerase (ThermoFisher, Waltham, MA) using 395

primer pairs LEJ_236 and LEJ_237. The 50 L PCR product was treated with 1.5 L DpnI (New 396

England Biolabs, Ipswich, MA) for 2 hours, transformed into DH5, and transformants were 397

plated onto LB agar containing kanamycin. Clones that contained the desired mutations were 398

confirmed by Sanger sequencing, plasmids were miniprepped and digested, and inserts were 399

subcloned into pPr01WH1266 to generate complementation vectors with desired point mutations. 400

H2O2 growth assays. LB was used as the growth medium for all assays and contained antibiotics 401

as necessary for maintenance of plasmids. Bacterial strains were freshly streaked to solid agar 402

and grown to stationary phase in 3 mL overnight cultures. Overnight cultures were sub-cultured 403

1:50 in LB for one hour prior to 1:100 inoculation of medium containing H2O2 (30%, EMD 404

Millipore, Darmstadt, DE) at the indicated concentrations. All growth assays were carried out in 405

96-well plates in 100 µL volume, and growth was measured by optical density absorbance at 600 406

nm. 407

H2O2 disc diffusion assays. Disc diffusion assays were modified from published protocols (68, 408

72). Freshly streaked strains were grown to stationary phase in 3 mL overnight cultures, diluted 409

1:10 in in LB, and 100 L of diluted culture was added to 4 mL of melted soft agar (0.75% agar) 410

and immediately poured onto LB agar plates, which were allowed to dry for 5 minutes. Sterile 6-411

mm paper discs were placed on the center of the plate and loaded with 10 L of 9.8 M H2O2 412


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(30% H2O2, EMD Millipore, Darmstadt, DE). After incubation at 37C for 20 hours, the zone of 413

growth inhibition was determined as the mean of three separate diameter measurements. 414

H2O2 killing assays. Biological triplicate overnight cultures of A. baumannii WT and the oxyR 415

strain were diluted 1:1000 into 10 mL LB in 50 mL conical tubes and grown to mid-exponential 416

phase. Cultures were treated with various concentrations of H2O2 (EMD Millipore, Darmstadt, 417

DE) ranging from 1 – 40 mM for 10 or 30 minutes. At each timepoint, 10 L of culture was 418

collected, serially diluted in PBS containing catalase (2,000 units/mL, catalase from bovine liver, 419

Sigma, St. Louis, MO) and spot-plated to LB agar for CFU enumeration. 420

OxyR protein alignment. The primary amino acid sequences of selected OxyR orthologs were 421

downloaded from ncbi.gov. Multiple sequence alignment was performed using ClustalOmega on 422

the EBI web server, which can be found at https://www.ebi.ac.uk/Tools/msa/clustalo/ (73). 423

OxyR phylogenetic tree. Phylogenetic tree analysis was performed on the Phylogeny.fr 424

platform (74). Sequences were aligned with MUSCLE (v3.8.31) using default settings (75). After 425

alignment, ambiguous regions were removed with Gblocks (v0.91b) (76) with the following 426

settings: minimum block length of 10, no gap positions allowed, maximum contiguous segment 427

of nonconserved positions of 8, and minimum number of sequences for a flank position of 85%. 428

The phylogenetic tree was reconstructed using the maximum likelihood method implemented in 429

the PhyML program (v3.1/3.0 aLRT) (77, 78) using the following settings: WAG model, aLRT 430

statistical test, 4 categories, estimated gamma and estimated invariable sites. Graphical 431

representation of the phylogenetic tree was performed with TreeDyn (v198.3) (79) using the 432

following settings: rectangular conformation, legend displayed, bootstrap branch annotation. 433

Purification of recombinant protein. The oxyR ORF was cloned into pET15b (Novagen, EMD 434

Millipore, Darmstadt, DE) to generate N-terminal hexahistidine-tagged constructs. The WT oxyR 435


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ORF was amplified by primer pair LEJ_227 and LEJ_228, double digested with NdeI and 436

BamHI-HF (New England Biolabs, Ipswich, MA), ligated into pET15b with T4 DNA ligase 437

(Promega, Madison, WI), and transformed into DH5. The OxyR C202S ORF was cloned by 438

PCR amplification of the ORF from miniprepped plasmid p.oxyR C202S using primer pairs 439

LEJ_227 and LEJ_228 and ligation into pCRBLUNT. Digestion and ligation were performed as 440

described for WT oxyR. E. coli BL21 (DE3) was transformed with the resultant vector and 441

grown at 37C to an OD600 of 0.4-0.6 before induction with 1 mM isopropyl -D-1-442

thiogalactopyranoxide (IPTG, Sigma, St. Louis, MO). Following induction, bacteria were 443

maintained at 30C for an addition 8-10 hours. Cells were harvested by centrifugation at 6000 x 444

g for 10 minutes, washed once with LB, and stored at -70C. Cells were thawed, resuspended in 445

lysis buffer [50 mM Tris pH 8, 300 mM NaCl, 20 mM imidazole, 1 mg/mL lysozyme, protease 446

inhibitor cocktail (Sigma, St. Louis, MO), and deoxyribonuclease (Sigma, St. Louis, MO)], 447

dounce homogenized, and lysed at 20,000 psi for 5 minutes in an EmulsiFlex homogenizer 448

(Aventin, Inc., Ottawa, ON). Following disruption, lysates were centrifuged at 15,000 rpm for 1 449

hour at 4C to remove the insoluble fraction. The supernatants were passed through a 45 micron 450

filter and applied to a Ni-NTA column (Qiagen, Hilden, DE). The column was washed with 20 451

bed volumes of wash buffer (50 mM Tris pH 8, 300 mM NaCl, 25 mM imidazole) followed by 452

sequential washes with increasing concentrations of imidazole (100 mM, 150 mM, 200 mM, 250 453

mM, 300 mM, 500 mM). OxyR eluted free of contaminating proteins at 200 mM imidazole. 454

Buffer exchange was performed using PD-10 desalting columns (GE Healthcare Life Sciences, 455

Little Chalfont, UK) to the final buffer (20 mM tris, pH 8, 500 mM NaCl, 5% glycerol, 10 mM 456

dithiothreitol). The OxyR C202S protein was purified using the same protocol as WT OxyR. 457


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Electromobility shift assays. Promoter DNA was amplified by PCR using primer pairs outlined 458

in Supplemental Table 1 and purified by Qiagen PCR clean up kit (Qiagen, Hilden, DE). Forty 459

ng of promoter was incubated for 30 minutes at room temperature with freshly purified OxyR or 460

OxyR C202S at the indicated concentrations in EMSA binding buffer (20 mM tris HCl pH 7.5, 461

2.5 mM MgCl2, 0.45 mM EDTA, 0.05% nonidet P-40, 10% glycerol, and 5 mM dithiothreitol) 462

that was modified with H2O2 as noted. Molarity of promoters in final solution was 13.5 nM for 463

A1S_1201 (ahpF1), 16.4 nM for A1S_3382 (katE), and 10 nM for mumT. Samples were 464

separated by electrophoresis in 6% polyacrylamide 0.5X TBE gels (5X TBE contained 89 mM 465

Tris base, 89 mM boric acid, and 1 mM EDTA) that were prerun at 100 V for 20 minutes. 466

Following electrophoresis, gels were stained with SYBR Green (Invitrogen, Carlsbad, CA) 467

diluted 1:10,000 in 0.5X TBE for 20 minutes in the dark, washed twice with H2O, and visualized 468

with a gel imager (BIO-RAD, Hercules, CA). 469

Mouse infections. A competitive infection murine model of pneumonia was utilized. Wild-type 470

A. baumannii and the isogenic, kanamycin-marked oxyR strain were freshly streaked from 471

frozen stocks onto LB agar or LB agar containing 40 μg mL-1

kanamycin, respectively, two days 472

prior to infection. Overnight cultures were grown in LB without antibiotic selection. On the day 473

of the infection, overnight cultures were sub-cultured 1:1,000 in 10 mL of LB and grown to mid-474

exponential phase. Cells were then harvested by centrifugation, washed twice in PBS, and 475

resuspended in PBS to a final concentration of 1 x 1010

CFU mL-1

. Suspensions of wild-type A. 476

baumannii and the oxyR strain were then combined in a 1:1 ratio, mixed thoroughly, and 477

immediately utilized for infection. Mice were anesthetized with intraperitoneal injection of 2,2,2-478

tribromoethanol diluted in PBS. Anesthetized mice were inoculated intranasally with 5 x 108 479

CFU in 45 microliter volume. Infection proceeded for 36 hours. Mice were then euthanized with 480


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CO2 and lungs and livers removed and placed on ice. Organs were homogenized in 1 mL PBS, 481

serially diluted in PBS with catalase (2,000 units/mL, catalase from bovine liver, Sigma, St. 482

Louis, MO), and dilutions were spot-plated onto LB agar and LB agar containing 40 μg mL-1

483

kanamycin. Isogenic mutant burdens were enumerated by counting colonies recovered on 484

kanamycin-containing plates. Infections were performed at the Vanderbilt University Medical 485

Center under the principles and guidelines described in the Guide for the Care and Use of 486

Laboratory Animals using Institutional Animal Care and Use Committee (IACUC)-approved 487

protocol M1600123-00. Vanderbilt University Medical Center is an American Association for 488

Laboratory Animal Science (AALAS)-accredited facility. The Vanderbilt University Medical 489

Center is registered with the Office of Laboratory Animal Welfare (OLAW), assurance number 490

A-3227-01. 491

Imaging of H2O2 production in vivo. Imaging was carried out similar to published methods 492

(35). FVB-luc+ (FVB-Tg(CAG-luc-GFP)L2G85Chco/J) mice were infected using the protocol 493

described above using WT A. baumannii. FVB-luc+ mice were purchased from Jackson 494

laboratories (Stock #008450) and were bred in-house at Vanderbilt University. After 36 hours of 495

infection, mice were injected intraperitoneally with 0.05 moles Peroxy Caged Luciferin-2 496

(PCL-2) and 0.05 moles D-cysteine in 50 L DMSO (Sigma, St. Louis, MO). Chemical probes 497

were provided by Chris Chang (UC Berkeley). Mice were imaged 30 minutes post-injection 498

using a Xenogen IVIS 200 (Caliper Life Sciences, Hopkinton, MA). Mice were anesthetized 499

prior to injection and during imaging via inhalation of isoflurane (Piramal, Bethlehem, PA). 500

Infections were performed at the Vanderbilt University Medical Center under the principles and 501

guidelines described in the Guide for the Care and Use of Laboratory Animals using Institutional 502

Animal Care and Use Committee (IACUC)-approved protocol M/14/243. 503


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Growth for RNASeq and RNA isolation. Biological triplicate overnight cultures of WT A. 504

baumannii and the oxyR strain were diluted 1:1000 into 10 mL LB in 50 mL conical tubes and 505

grown to mid-exponential phase. Cultures were treated with 5.65 L of 30% H2O2 (EMD 506

Millipore, Darmstadt, DE), for a final concentration of 5 mM, or untreated at 37C with shaking 507

at 180 rpm for 10 minutes. Following treatment, 10 L of bacterial culture was saved for CFU 508

plating, while the remaining bacterial culture was immediately mixed with 10 mL 1:1 mixture of 509

ice-cold acetone (JT Baker, Center Valley, PA) and ethanol (Sigma, St. Louis, MO) and frozen at 510

-70C. CFU were enumerated by serially diluting cultures in PBS containing catalase (2,000 511

units/mL, catalase from bovine liver, Sigma, St. Louis, MO) and spot-plating to LB agar. For 512

RNA purification, thawed cell suspensions were centrifuged at 7,000 rpm for 10 minutes, 513

supernatants were decanted, and pellets were dried on paper towels. Pellets were resuspended in 514

LETS buffer (0.1 M LiCl, 10 mM EDTA, 10 mM Tris HCl pH 7.4, 1% SDS), homogenized in a 515

bead beater (Fastprep-24, MP Biomedical, Santa Ana, CA) with Lysing Matrix B beads (MP 516

Biomedicals, MP Biomedical, Santa Ana, CA) at a speed of 6 m/s for 45 seconds, heated at 55C 517

for 5 minutes, and centrifuged for 10 minutes at 15,000 rpm. The upper phase was collected, 518

mixed with 1 mL TRI Reagent (Sigma, St. Louis, MO), and incubated for 5 minutes at room 519

temperature. Chloroform (0.2 mL, Acros Organics, Waltham, MA) was added, samples were 520

vigorously shaken for 15 seconds, and samples were incubated at room temperature for 2 521

minutes. Following centrifugation at 4C for 15 minutes, 600 L of the upper aqueous phase was 522

collected and RNA was precipitated with 1 mL isopropanol (Sigma, St. Louis, MO). RNA was 523

washed with 70% ethanol (Sigma, St. Louis, MO) and resuspended in 100 L DNase-Free, 524

RNase-Free water (ThermoFisher, Waltham, MA). DNA contamination was removed by 525

treatment with 8 L RQ1 enzyme (Promega, Madison, WI), 12 L 10X RQ1 buffer, and 2 L 526


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Ribolock RNase inhibitor (ThermoFisher, Waltham, MA) for 2 hours at 37C. DNase was 527

removed and samples further purified by the RNEasy kit (Qiagen, Hilden, DE) using the 528

manufacturer’s instructions and RNA was stored long-term at -80C. Each biological replicate 529

was submitted for sequencing without technical replicates. 530

RNA-seq library preparation and sequencing. RNA-seq library construction and sequencing 531

was performed by HudsonAlpha (Huntsville, AL). The concentration and integrity of the total 532

RNA was estimated by Qubit® 2.0 Fluorometer (Invitrogen, Carlsbad, California), and Agilent 533

2100 Bioanalyzer (Applied Biosystems, Carlsbad, CA), respectively. Five hundred ng of total 534

RNA was required for proceeding to downstream RNA-seq applications. First, ribosomal RNA 535

(rRNA) was removed using Ribo-Zero™ Gold (Epidemiology) kit (Illumina, San Diego, CA) 536

using manufacturer's recommended protocol. Immediately after the rRNA removal, the RNA 537

was fragmented and primed for the first strand synthesis using the NEBNext® First Strand 538

synthesis module (New England BioLabs Inc., Ipswich, MA). Directional second strand 539

synthesis was performed using NEBNext® Ultra Directional second strand synthesis kit. 540

Following this, the samples underwent standard library preparation protocol using NEBNext® 541

DNA Library Prep Master Mix Set for Illumina® with slight modifications. Briefly, end-repair 542

was done followed by polyA addition and custom adapter ligation. Post-ligated materials 543

were individually barcoded with unique in-house Genomic Services Lab (GSL) primers and 544

amplified through 12 cycles of PCR. Library quantity was assessed by Qubit 2.0 Fluorometer, 545

and the library quality was estimated by utilizing a DNA High Sense chip on a Caliper Gx 546

(Perkin Elmer). Accurate quantification of the final libraries for sequencing applications was 547

determined using the qPCR-based KAPA Biosystems Library Quantification kit (Kapa 548

Biosystems, Inc., Woburn, MA). Each library was diluted to a final concentration of 12.5 nM 549


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and pooled equimolar prior to clustering. Paired-End (PE) sequencing was performed on an 550

Illumina HiSeq2500 sequencer (Illumina, Inc.). Raw RNA sequencing data and processed data 551

are deposited in the NCBI GEO under accession GSE114130. 552

Processing of RNA-seq reads. RNA-seq analysis was performed by HudsonAlpha (Huntsville, 553

AL). Approximately 25 million, 100bp, PE reads were generated from each sample. Further 554

downstream analysis of the sequenced reads from each sample was performed as per the Hudson 555

Alpha unique in-house pipeline. Briefly, quality control checks on raw sequence data from each 556

sample were performed using FastQC (Babraham Bioinformatics, London, UK). Raw reads were 557

imported onto the commercial data analysis platform, Avadis NGS (Strand Scientifics, CA, 558

USA) and mapped to the reference genome A. baumannii ATCC17978. After quality inspection, 559

the aligned reads were filtered on the basis of read quality metrics where reads with a base 560

quality score less than 30, alignment score less than 95, and mapping quality less than 40 were 561

removed. Remaining reads were then filtered on the basis of their read statistics, where missing 562

mates, translocated, unaligned and flipped reads were removed. The reads list was then filtered 563

to remove duplicates. Samples were grouped and quantification of transcript abundance was 564

done on this final read list using Trimmed Means of M-values (TMM) (80) as the normalization 565

method. Differential expression of genes was calculated on the basis of fold change (using 566

default cut-off ≥ ±2.0) observed between defined conditions, and the P-value of the differentially 567

expressed gene list was estimated by Z-score calculations using determined by Benjamini 568

Hochberg FDR correction of 0.05 (81). 569

Quantitative RT-PCR. Two g of RNA was reverse transcribed by M-MLV reverse 570

transcriptase (Fisher Scientific, Waltham, MA) in the presence of random hexamers (Promega, 571

Madison, WI) and Ribolock RNase inhibitor (ThermoFisher, Waltham, MA). Reactions 572


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containing no reverse transcriptase were used to control for DNA contamination. Resultant 573

cDNA was diluted 1:100 and subjected to qRT-PCR using iQ SYBR green supermix (BIO-RAD, 574

Hercules, CA) with primer pairs listed in Supplemental Table 1. Amplification was performed 575

on a CFX96 qPCR cycler (Bio-Rad, Hercules, CA) using a 3-step melt curve program. CT 576

values for each transcript were normalized by 16s and fold changes were calculated using the 577

CT method. 578

Statistical analyses. All raw numerical data were saved in Excel files and imported into 579

GraphPad Prism for statistical analysis. Specific statistical tests employed each experiment are 580

outlined in the figure legends. 581

Availability of data and materials. Any materials and data will be made available to members 582

of the scientific community upon communication with the Corresponding Author. 583

584

Acknowledgments 585

L.J.J. conceived of the project, performed experiments, and wrote the manuscript. E.P.S. 586

supervised all studies and helped to write the manuscript. E.R.G. carried out EMSA experiments. 587

Z.R.L. performed the bioluminescent experiment. C.J.C. and M.C.H. provided the 588

bioluminescent probe. We thank members of the Skaar laboratory for reviewing the manuscript 589

and Joe Zackular for uploading the RNASeq data to GEO. Work in E.P.S.’s laboratory was 590

supported by Public Health Service grants AI101171, AI069233, and AI073843 and the Defense 591

Advanced Research Projects Agency (DARPA). L.J.J. was supported by American Heart 592

Association grant 15PRE25060007 and Public Health Service award T32 GM07347 from the 593

National Institute of General Medical Studies for the Vanderbilt Medical-Scientist Training 594

Program. L.J.J. is a P.E.O. Scholar. E.R.G. was supported by training grant T32HL094296. 595


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Z.R.L. was supported by T32 ES007028 and F31 AI136255. We thank the NIH for grant support 596

of C.J.C. (GM79465). C.J.C. is an Investigator with the Howard Hughes Medical Institute. 597

M.C.H. thanks the University of California President’s Postdoctoral Program for post-doctoral 598

fellowship support. 599

Conflict of interest statement: The authors have declared that no conflicts of interest exist. 600


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82. Hood MI, Mortensen BL, Moore JL, Zhang Y, Kehl-Fie TE, Sugitani N, Chazin WJ, 825

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PLOS Pathog 8:e1003068. 828

829


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Figure Legends 830

Figure 1. H2O2 is produced during A. baumannii lung infection. (A) Schematic of 831

experimental design. (B-C) Mice were infected with A. baumannii WT or mock-infected with 832

PBS. Imaging was performed 36 hours post infection. Production of H2O2 was monitored in vivo 833

by injection of a caged luciferin probe (PCL-2) that specifically reacts with H2O2 to generate 834

bioluminescence (35). Mice were placed in the prone position (B) and in the supine position (C). 835

The results are representative of two independent experiments. 836

Figure 2. H2O2 exposure alters the transcriptome of A. baumannii in an oxyR-dependent 837

manner. RNA sequencing was performed on RNA extracted from WT A. baumannii and the 838

oxyR strain grown to mid-exponential phase and treated with 5 mM H2O2 for 10 minutes. (A) 839

Classification of transcript IDs significantly up- or down-regulated in WT A. baumannii treated 840

with H2O2 compared to untreated cells (>2 log2 fold change; P < 0.05 corrected for FDR). (B) 841

Venn diagram comparing common upregulated transcripts in the following conditions: WT + 842

H2O2 (relative to WT untreated), oxyR strain + H2O2 (relative to oxyR untreated), and oxyR 843

strain untreated (relative to WT untreated). (C) Validation of selected RNA sequencing results 844

by qRT-PCR in WT A. baumannii and the oxyR strain treated with H2O2. Results are depicted 845

as fold-change of gene transcript abundance following 10-minute treatment with 5 mM H2O2 846

relative to no treatment (untx). FC = fold change. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by t-847

test against theoretical value of 1.0. #, P < 0.05; ##, P < 0.01; ###, P < 0.001 by t-test comparing 848

WT and the oxyR strain. N = 3 biological replicates, mean +/- SD. 849

Figure 3. Inactivation of oxyR impairs A. baumannii growth in the presence of H2O2. 850

Growth of WT A. baumannii and the oxyR strain was monitored under various conditions (A-851

D). (A) Growth of A. baumannii in LB containing 100 M H2O2 was monitored by OD600. (B) 852


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Inhibition of growth by H2O2 at the indicated concentrations at the six hour timepoint. Growth as 853

a percent of untreated was calculated by OD600. For (A) and (B), growth curves are from a single 854

experiment performed in biological triplicate and are representative of three independent 855

experiments. (C) Zone of inhibition by 10 L of 9.8 M H2O2 spotted onto discs placed on solid 856

medium. Representative plates are shown after overnight growth. (D) Quantification of the 857

diameter of inhibition by H2O2. Data are combined from three independent experiments with 858

three biological replicates in each experiment. ***, P < 0.001 by Student’s t-test. 859

Figure 4. A. baumannii OxyR contains a conserved cysteine residue that is important for 860

function. (A) Phylogenetic tree based on protein sequence alignment of A. baumannii and 861

Acinetobacter baylyi OxyR with experimentally characterized OxyR proteins in other Gram-862

negative organisms. Branch length is representative of sequence divergence. (B) Amino acid 863

sequence alignment for a portion of the substrate-binding domain of OxyR that contains 864

conserved cysteine residues. Asterisk indicates complete conservation, one dot indicates similar 865

residues at that position, and two dots indicate highly similar residues at that position. The amino 866

acid positions highlighted with a rectangle contain a cysteine in the A. baumannii OxyR 867

sequence. (C) Growth of WT A. baumannii with empty vector, the oxyR strain with empty 868

vector, the oxyR strain expressing WT oxyR in trans, or the oxyR strain expressing oxyR 869

C202S in trans in 30 µM H2O2. The growth curve is from a single experiment performed in 870

biological triplicate and is representative of three independent experiments. 871

Figure 5. A. baumannii inactivated for oxyR is pre-adapted to H2O2 stress during 872

exponential growth. (A) WT A. baumannii and oxyR strain mid-exponential phase cultures 873

were treated with bolus doses of H2O2 at the indicated concentrations for 30 minutes. CFU 874

enumerated following bolus exposure are shown on a log10 scale. The limit of detection was 2-875


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log10. *, P < 0.05; ***, P < 0.001 by t-test. Bars indicate mean +/- SD. (B) qRT-PCR 876

quantification of relative abundance of H2O2-responsive transcripts in WT A. baumannii and 877

oxyR strain in LB alone. FC = fold change. *, P < 0.05; **, P < 0.01 by t-test against theoretical 878

value of 1.0. N = 6 biological replicates, mean +/- SD. (C) A. baumannii WT and oxyR strain 879

mid-exponential phase cultures were pre-treated with 1 mM H2O2 or vehicle for 30 minutes. 880

Following pre-treatment, cultures were exposed to a bolus of 60 mM H2O2 for 10 minutes. CFU 881

enumerated following bolus exposure are shown on a log10 scale with the limit of detection at 2-882

log10. *, P < 0.05; ***, P < 0.001 by one-way ANOVA with Tukey’s multiple comparisons test. 883

Bars indicate mean +/- SD. For (A-B), data are from a single experiment performed in biological 884

triplicate and are representative of five independent experiments. 885

Figure 6. OxyR binds the promoters of ahp and kat. PCR amplified promoter regions for (A) 886

A1S_1201 (ahpF1) and (B) A1S_3382 (katE) were incubated with purified recombinant His6-887

OxyR alone (A, B) or WT His6-OxyR or His6-OxyR C202S (C, D). The protein was 888

preincubated for 10 minutes with or without 50 mM H2O2. DNA was visualized using SYBR 889

Green. Each gel is representative of experiments performed on at least two separate days. 890

Figure 7. oxyR promotes the fitness of A. baumannii in a murine model of pneumonia. 891

Bacterial burdens were enumerated from the lungs (A) and livers (B) of mice 36 hours following 892

intranasal inoculation of a 1:1 mixture of WT A. baumannii and oxyR. (A) Lung CFU. Data are 893

from one experiment. N = 10. (B) Liver CFU. Data are combined from two independent 894

experiments. N = 25. ***, P < 0.001 by Mann-Whitney. 895

896


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Tables 897

Table 1: Bacterial strains used in this study. 898

Table 2: Genes with increased expression in WT A. baumannii following treatment with 899

H2O2. 900

Table 3: Genes with increased expression in oxyR strain following treatment with H2O2. 901

Table 4: Genes with increased expression in the oxyR strain in LB. 902


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Table 1: Bacterial strains used in this study

Strain Relevant characteristics Reference

or source

17978 Wild-type Acinetobacter baumannii ATCC

ΔoxyR In-frame ΔoxyR::aphA This study

17978 pWH1266 Empty vector control with plasmid

pPr01WH1266

(82)

ΔoxyR pWH1266 Empty vector control with plasmid

pPr01WH1266

This study

ΔoxyR poxyRWH1266 Complementation strain containing

plasmid p.Pr01.oxyR.WH1266

This study

ΔoxyR poxyR C202S

WH1266

Complementation strain containing

plasmid p.Pr01.oxyR.WH1266 with

C202S mutation

This study

17978 pAT02 WT harboring recAb machinery for

recombineering

(33)

Escherichia coli DH5

p.oxyR

Cloning strain containing plasmid

p.Pr01.oxyR.WH1266

This study


p.oxyR C202S

Cloning strain containing plasmid

p.Pr01.oxyR.WH1266 with C202S

mutation

This study


p.oxyR pCRBlunt

Cloning strain containing oxyR in

pCRBlunt

This study


p.oxyR C202S

pCRBlunt

Cloning strain containing oxyR with

C202S mutation in pCRBlunt

This study

Escherichia coli BL21

(DE3) p.oxyR.ET15B

Protein expressing strain for oxyR

cloned into pET15b

This study


p.oxyR.ET15B

Cloning strain for oxyR cloned into

pET15b

This study

Escherichia coli BL21

(DE3)

p.oxyRC202S.ET15B

Protein expressing strain for OxyR

C202S cloned into pET15b

This study


p.oxyRC202S.ET15B

Cloning strain for oxyR C202S cloned

into pET15b

This study

Acinetobacter

baumannii AB5075

Wild-type parental strain for transposon

library

(38)

AB07598 AB5075 transposon mutant

ABUW_2905-184::T26

(38)


ABUW_2905-143::T26

(38)


ABUW_2905-146::T26

(38)


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ABUW_2905-156::T26

(38)


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Table 2: Genes with increased expression in WT A. baumannii following treatment with

H2O2.

Locus Annotation (KEGG) Fold change

Peroxide detoxification

A1S_1459 alkyl hydroperoxide reductase subunit F ahpF2 61.9





A1S_3382 Catalase katE 27.7

A1S_2863 putative antioxidant protein 5.9

Sulfur homeostasis

A1S_2533 putative esterase 459.7

A1S_2532 sulfate transport protein 364.5

A1S_2531 sulfate transport system substrate-binding protein 334.4

A1S_2534 sulfate transport system permease protein 172.9

A1S_0030 sulfonate transport system substrate-binding protein 165.8

A1S_0028 alkanesulfonate monooxygenase 113.7

A1S_2535 sulfate transport system permease protein 106.1

A1S_0029 sulfonate transport system substrate-binding protein 71.2

A1S_0027 sulfonate transport system permease protein 49.6


A1S_1408 putative rhodanese-related sulfurtransferase 25.5

A1S_2536 sulfate transport system ATP-binding protein 18.1

A1S_0977 arylsulfatase 18.0

A1S_3306 dimethylsulfone monooxygenase 17.9

A1S_0026 sulfonate transport system ATP-binding protein 16.2

A1S_2846 sulfite reductase (NADPH) hemoprotein beta-component 5.6

Iron homeostasis

A1S_2389 iron complex transport system permease protein 26.2

A1S_2381 2,3-dihydroxybenzoate-AMP ligase 21.8

A1S_1719 4Fe-4S ferredoxin iron-sulfur binding 21.4

A1S_2380 bifunctional isochorismate lyase 18.2

A1S_2379 histidine decarboxylase 16.3

A1S_2382 BasD 16.0

A1S_2388 iron complex transport system permease protein 15.8

A1S_2387 iron complex transport system ATP-binding protein 14.9

A1S_2390 putative acinetobactin biosynthesis protein 8.7

A1S_2372 isochorismate synthase 8.2

A1S_2386 iron complex transport system substrate-binding protein 7.4

A1S_2383 putative acinetobactin biosynthesis protein 6.9

A1S_2392 putative acinetobactin utilization protein 6.1

A1S_2077 putative outer membrane porin receptor for Fe(III)-coprogen, 5.7


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Fe(III)-ferrioxamine B and Fe(III)-rhodotrulic acid uptake (fhuE)

A1S_3339 iron complex outermembrane receptor protein 5.4

A1S_1607 iron complex outermembrane receptor protein 4.6

A1S_1647 putative siderophore biosynthesis protein 4.4

A1S_1359 iron(III) transport system substrate-binding protein 4.3

A1S_2530 4-amino-4-deoxychorismate lyase 4.1

A1S_1634 Rrf2 family transcriptional regulator 4.1

Phosphate transport

A1S_2448 phosphate transport system substrate-binding protein 10.5

A1S_2447 phosphate transport system permease protein 6.8

A1S_3374 positive pho regulon response regulator 6.0

A1S_3376 phosphate regulon sensor histidine kinase PhoR 4.7

A1S_0256 phosphate transport system protein 4.4

Nucleic acid synthesis and repair

A1S_2273 RNA 3'-terminal phosphate cyclase (ATP) 26.4

A1S_2586 deoxyguanosinetriphosphate triphosphohydrolase 8.6

A1S_0310 excinuclease ABC subunit C 6.4

A1S_1173 DNA polymerase V 5.3




A1S_1962 recombination protein RecA 4.4

A1S_3295 excinuclease ABC subunit A 4.2

A1S_3359 topoisomerase IV subunit B 4.1

Amino acid transport and metabolism

A1S_1443 taurine transport system ATP-binding protein 222.8

A1S_0023 putative malic acid transport protein 135.0

A1S_1397 polar amino acid transport system permease protein 102.2

A1S_1444 taurine transport system permease protein 87.3

A1S_1442 taurine transport system substrate-binding protein 85.9

A1S_1396 polar amino acid transport system permease protein 76.5

A1S_1445 taurine dioxygenase 41.8

A1S_1398 polar amino acid transport system ATP-binding protein 27.1

A1S_0921 arginine:ornithine antiporter / lysine permease 14.7

A1S_1399 polar amino acid transport system substrate-binding protein 11.4

A1S_1485 D-methionine transport system substrate-binding protein 9.9

A1S_2384 lysine N6-hydroxylase 6.6

A1S_1046 lysine exporter 6.3

A1S_1407 serine O-acetyltransferase 6.1

A1S_1400 polar amino acid transport system substrate-binding protein 4.8

Ribosome

A1S_2271 tRNA-splicing ligase RtcB 118.6

A1S_1961 ribosome-associated heat shock protein Hsp15 5.1

Fatty acid metabolism

A1S_1436 putative acyl-CoA dehydrogenase 7.8


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A1S_2458 linoleoyl-CoA desaturase 7.5


Transport

A1S_1456 chromate transporter 23.1

A1S_1457 chromate transporter 13.1

A1S_2378 putative ABC transporter 8.4

A1S_1720 NitT/TauT family transport system substrate-binding protein 8.2

A1S_3272

MFS transporter, YNFM family, putative membrane transport

protein 5.7

A1S_2377 putative ABC transporter 5.4

A1S_2311 ABC-2 type transport system ATP-binding protein 4.4

A1S_1772 MFS transporter, DHA2 family, multidrug resistance protein 4.2

Metabolism (other)

A1S_1488 putative Acyl-CoA dehydrogenase 100.9

A1S_1487 putative Acyl-CoA dehydrogenase 30.0

A1S_3305 FMN reductase 24.6

A1S_1486 putative monooxygenase (DszA-like) 24.5

A1S_0922 putative homocysteine S-methyltransferase family protein 19.9


A1S_0024 adenosylhomocysteine nucleosidase 10.9

A1S_0463 putative alkaline phosphatase 9.0

A1S_2459 putative oxidoreductase 8.3

A1S_3222 homocysteine synthase 6.9

A1S_2293 ferredoxin/flavodoxin---NADP+ reductase 5.5

A1S_1446 allantoin racemase 4.4

A1S_0131 streptomycin 3"-adenylyltransferase 4.3

Other

A1S_1717 GntR family transcriptional regulator 32.1

A1S_1414 LrgB-like protein 27.5

A1S_0466 sec-independent protein translocase protein TatA 25.0

A1S_1665 putative membrane protein 19.9

A1S_3114 CBS domain-containing membrane protein 19.0

A1S_1677 putative porin precursor 17.5

A1S_3381 AnkB protein 17.4

A1S_0465 sec-independent protein translocase protein TatB 16.1

A1S_1963 regulatory protein 13.2

A1S_0464 sec-independent translocation TatC 12.8

A1S_1928 putative signal peptide 12.4

A1S_2473 transcriptional regulator LysR family 10.4

A1S_1224 transposase 8.9

A1S_0416 putative transcriptional regulator (LysR family) 5.9


A1S_1438

putative coenzyme F420-dependent N5N10-methylene

tetrahydromethanopterin reductase 5.2


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A1S_3375 phosphate regulon response regulator PhoB 5.2

A1S_1539 ArsR family transcriptional regulator 4.7

A1S_1767 putative acid phosphatase 4.7

A1S_1503 transmembrane pair 4.5




A1S_1197 putative extracellular nuclease 4.1

Hypothetical

A1S_0517 hypothetical protein 139.3
































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Table 3: Genes with increased expression in oxyR strain following treatment with H2O2.

Locus Annotation Fold change

Nucleic acid synthesis and repair



Transport

A1S_3272

MFS transporter, YNFM family, putative

membrane transport protein 7.8

Metabolism (other)

A1S_0567 NAD(P) transhydrogenase subunit alpha 12.4

A1S_0566 NAD(P) transhydrogenase subunit alpha 11.3

A1S_0568 NAD(P) transhydrogenase subunit beta 9.8

Hypothetical










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Table 4: Genes with increased expression in the oxyR strain in LB

Locus Annotation (KEGG) Fold change

Peroxide detoxification







Sulfur homeostasis



Iron homeostasis

A1S_1647 putative siderophore biosynthesis protein 4.4

Amino acid transport and metabolism

A1S_1528 hypothetical 24.0

A1S_3046 oligopeptidase A 5.2

A1S_3047 oligopeptidase A 5.1

Chaperones

A1S_2959 molecular chaperone GrpE 5.1

A1S_2960 molecular chaperone DnaK 4.6

A1S_2665 K04078 chaperonin GroES 4.4

Metabolism (other)

A1S_0804 trehalose 6-phosphate phosphatase 4.6

Other

A1S_0646 intracellular multiplication protein IcmB 4.5

A1S_0647 intracellular multiplication protein IcmO 4.5

Hypothetical




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